Trypanosoma cruzi is a single-cell protozoan parasite that causes Chagas disease, characterized by cardiac or gastrointestinal complications in chronically infected patients. There is no curative treatment yet available and new-drug targets against the parasite are urgently needed. T. cruzi presents a complex life- cycle involving a Triatomine insect vector and several possible vertebrate hosts including humans. Parasite surface components are suspected to play essential roles to fulfill survival strategies in their sequential host environments. Indirect experimental evidences suggest that surface glycoinositolphospholipids (GIPLs) and mucin-type glycoproteins protect the parasites from proteases, complement, lytic antibodies, phagocytosis, and function as potential immunomodulators that could even determine the fate of pathogenesis during infection. Exclusive features of mucin-type glycoproteins include the sugar 1GlcNAc forming the bridge between O-glycans and Thr residues of the mucins, 2Gal residues found both in the pyranosidic and/or furanosidic forms, and terminal 13Gal or sialic-acid residues, the later being transferred from host glycoconjugate donors to the parasite's surface Gal acceptors by the action of parasite cell surface trans- sialidases. Our goal is to determine if mucin-type O-glycosylation works as virulence factor and help the parasite to survive and succeed in its different environments during its life-cycle. We hypothesize that the Golgi enzymes UDP-GlcNAc:polypeptide N-acetylglucosaminyl transferase (pp-1GlcNAcT) and UDP- Gal:galactoside 13-galactosyltransferase (13GalT), respectively responsible for the addition of the first 1GlcNAc to Thr and the terminal 13Gal residues in the mucin-type O-glycans, are essential for the correct assembly of T. cruzi surface architecture. Two specific aims are proposed to test this. In Aim 1, conditional double knock-outs for each of the three TcOGNT genes (1, 2 and L) that encode pp-1GlcNAcTs will be obtained by targeted gene replacement, the three TcOGNT homologs will be expressed alone or in combination in Leishmania tarentolae, a related non-pathogenic trypanosomatid, and T. cruzi, and expression of the TcOGNTs will be mapped over serial life cycle stages. The genetically modified parasites will be used to evaluate defects in glycosylation, enzyme activities and on cell function, including viability, capacity to colonize invertebrate hosts, differentiate, resist to lysis by the alternative pathway of complement, and/or to adhere, infect and survive inside mammalian host cells. In Aim 2, the putative Golgi 13GalT gene of T. cruzi will be analyzed, by testing its predicted 13GalT activity after heterologous expression in Dictyostelium, and investigating its biochemical and cellular function by targeted gene disruption and over-expression in T. cruzi. This project is focused on basic questions about the biochemical and cellular functions of the putative glycosyltransferases, but future work is expected to investigate their roles in virulence in pursuit of new drug targets for treatment of Chagas disease.